1. Field of the Invention
The present invention relates to an epi-illumination microscope for use in observing light emitted from a sample.
2. Description of the Related Art
In general, a fluorescence observing epi-illumination microscope has been used for detecting fluorescence-labeled protein, tissue, gene and the like on a biological tissue cell in a biological or medical field. Especially, in recent years, a research of a micro sample having a level of one molecule that emits only feeble fluorescence, or a research using a living sample, not a fixed sample has been vigorously performed. Additionally, it is possible to develop light-emitting protein in the cell, detection or analysis has been possible while more physiological activity is kept. In a case where this living sample is observed, it has been demanded that fluorescence generated from the sample be efficiently detected with less irradiation energy while reducing noises entering an observation system as much as possible in order to reduce damages on the sample and correctly observe or analyze the sample. The feeble fluorescence having one molecule level cannot be detected until the noises that enter the observation system are minimized.
As one generation source of the noise, in general, there is a fluorescence filter set. FIG. 21 shows a constitution of a conventional microscope comprising the fluorescence filter set. As shown in FIG. 21, a fluorescence filter set 500 comprises three optical elements including an excitation filter 510, a light splitter 520, and an absorption filter 530. The excitation filter 510 selects only light having a predetermined wavelength from light from a light source 10 located off an optical axis of an optical observation system including an objective lens 30, an image forming lens 50, and a detection device 60. The light splitter 520 reflects the light having the wavelength selected by the excitation filter 510 to epi-illuminate a sample 40, and transmits the fluorescence generated from the sample 40 to guide it to the detection device 60. The light splitter 520 comprises a transparent substrate having a flat plate shape, the front surface of the transparent substrate is dichroic-mirror-coated, and the back surface thereof is coated with a reflection preventive film. The light splitter 520 is disposed with a tilt of 45 degrees with respect to an optical observation axis. The absorption filter 530 selectively excites the fluorescence transmitted through the light splitter 520, and cuts the wavelength light selected by the excitation filter 510. In general, the fluorescence filter set 500 is prepared for each wavelength of a fluorescent dyestuff.
In general, each of these optical elements (excitation filter 510, light splitter 520, absorption filter 530) comprises an interference filter whose parallel flat plate is coated with an interference film. Assuming that a maximum transmission wavelength is λ, an optical length of a dielectric is t, and a refractive angle in a boundary is φ, an interference condition of the interference film is represented by 2t·cos φ=mλ.
Here, assuming that an order m is constant and an interference condition is constant, the wavelength λ is proportional to cos φ. Although φ denotes the refractive angle, and is brought into a conjugated relation with respect to an incident angle by Snell's law, and both the angles are considered to be equal. Therefore, when the incident angle increases in the above equation, cos φ decreases, the wavelength λ also decreases, and a maximum transmittance portion gradually shifts to a short wavelength side. Therefore, when vertical incidence changes to oblique incidence, and the incident angle with respect to the interference film increases, a band opposite to a transmission band gradually shifts to the short wavelength side.
In the fluorescence filter set, the excitation filter and the absorption filter are designed to be optimum with respect to vertical incidence, and the light splitter is designed to be optimum with respect to 45 degree incidence. When the incident angle is different from a designed value, the wavelength light reflected at the incident angle having the designed value is transmitted, and the wavelength light transmitted at the incident angle having the designed value is reflected.
To perform efficient fluorescence observation, the light splitter may preferably completely reflect light of a transmission wavelength band of the excitation filter (first selection member), and completely transmit the light of a transmission wavelength band of the absorption filter (second selection member). However, a peak of an excitation wavelength of the fluorescent dyestuff, and that of an emission wavelength are near 10 to 20 mm in many cases. In general, a transmission band of the excitation filter (first selection member) can be brought close to that of the absorption filter (second selection member) to such an extent that the bands do not overlap with each other. However, when the light splitter is disposed at 45 degrees with respect to the optical axis, the light is separated with PS polarization. Therefore, unlike the transmission bands of the excitation filter (first selection member) and the absorption filter (second selection member), there is a limitation on thin-film design in bringing the transmission wavelength band close to a reflection wavelength band.
Moreover, when the band of the excitation wavelength of the fluorescent dyestuff is broad, the transmission wavelength band of the excitation filter (first selection member) is to be sometimes broadened. However, a conventional light splitter has a constitution in which one face of the transparent substrate is coated with a dichroic mirror having an only stacked portion for mainly reflecting the light transmitted through the excitation filter (first selection member), a film constitution is only changed, and there is a limitation to the broadening of the reflection band.
Exciting light is undesirably permitted to pass through the light splitter with respect to a certain wavelength band by these two factors. Therefore, the light that is not reflected by and is transmitted through the light splitter strikes on a side wall face of the fluorescence filter set, and is scattered by the side wall face. When this light ray enters the absorption filter at an angle deviating from the vertical incidence, even the light on a short wavelength side passes through the absorption filter unlike the vertical incidence, and this generates the noise. The light that has entered the side wall face of the fluorescence filter set emits self fluorescence, and even the self fluorescence emitted on the side of the absorption filter passes through the absorption filter.
Additionally, there is scattered light or self fluorescence generated when the excitation filter is irradiated with intense and broad-band light from the light source. A part of the scattered light or self fluorescence is emitted toward the light splitter or the absorption filter, but the incident angle of the light upon the light splitter approaches the vertical incidence from 45 device incidence indicating the designed value, and the incident angle upon the absorption filter turns to the oblique incidence from the vertical incidence indicating the designed value. That is, the reflection band of the light splitter shifts toward a long wavelength side, and the transmission band of the absorption filter shifts toward a short wavelength side. As a result, the scattered light or self fluorescence other than the light of the transmission band shifted toward the short wavelength side of the absorption filter and the long wavelength side of the light splitter passes through the light splitter or the absorption filter to generate the noise.